Compounds and pharmaceutical compositions containing them

ABSTRACT

Aminophosphonates alpha substituted by phenol groups of formula (I) have lipoprotein(a) lowering activity.

This application is a division of Ser. No. 08/973/669 filed Apr. 3,1998, now U.S. Pat. No. 6,060,464 which is a 371 of InternationalApplication No. PCT/EP96/02842 filed Jun. 26, 1996.

This invention relates to a new therapeutic use of aminophosphonatecompounds for lowering plasma and tissue levels of lipoprotein(a). Inparticular, this invention provides a new use of aminophosphonatederivatives, for the preparation of pharmaceutical compositions usefulin the treatment of diseases or disorders associated with high plasmaand tissue concentrations of lipoprotein(a); such as, for instanceartherosclerosis, thrombosis, restenosis after angioplasty and stroke.This invention also provides a method for increasing thrombolysis andpreventing thrombosis and a method of treatment of restenosis afterangioplasty by administering to a patient in need thereof anaminophosphonate compound at a dose effective for lowering plasma andtissue lipoprotein(a) levels. In addition, this invention also providesa group of new aminophosphonate compounds for use in the above mentioneduses and compositions.

Recent epidemiologic studies have shown a strong association betweenelevated lipoprotein(a) [Lp(a)] plasma levels and the occurrence ofcoronary heart disease, stroke and peripheral artery disease. Lp(a) isnow recognized as an independent risk factor for cardiovasculardiseases; in addition its role in promoting thrombosis by decreasingthrombolysis is increasingly acknowledged, see for instance“Lipoprotein(a) as A Risk Factor for Preclinical Atherosclerosis” P. J.Schreiner, J. D. Morrisett, A. R. Sharrett, W. Patsch, H. A. Tyroler, K.Wu and G. Heiss; Arteriosclerosis and Thrombosis 13 p. 826-833 (1993);“Detection and Quantification of Lipoprotein(a) in the Arterial Wall of107 Coronary Bypass Patients” M. Rath, A. Niendorf, T. Reblin, M.Dietel, H. J. Krebber and U. Beisiegel; Arteriosclerosis 9, p. 579-592(1989); and “Lipoprotein(a): Structure, Properties and PossibleInvolvement in Thrombogenesis and Atherogenesis” A. D. MBewu and P. N.Durrington; Atherosclerosis 85, p. 1-14 (1990). The potential ofthrombosis involvement in vessel occlusion and acute cardiovascularsyndrome is being increasingly recognized. One of the mechanisms thatmediate thrombosis associated with atherosclerotic plaque ruptureinvolves elevated levels of lipoprotein(a). The structure of Lp(a)consists of a low-density lipoprotein (LDL)-like particle with aglycoprotein, apolipoprotein(a) [apo(a)] that is linked via a disulfidebridge to the apo B-100 moiety of the LDL. Structurally there isstriking analogy between apo(a) and plasminogen, the precursor ofplasmin which cleaves fibrin to dissolve blood clots. However, unlikeplasminogen apo(a) is not a substrate for plasminogen activators. Thisstructural resemblance has led researchers to postulate and laterdemonstrate that apo(a) interferes with the normal physiologicalfunction of plasminogen, leading to a potential thrombogenic activity ofLp(a) see for instance:

“Activation of Transforming Growth Factor-β is Inversely Correlated withThree Major Risk Factors for Coronary Artery Disease: Lipoprotein(a),LDL-Cholesterol and Plasminogen Activator Inhibitor-1”, A. Chauhan, N.R. Williams, J. C. Metcalfe, A. A. Grace, A. C. Liu, R. M. Lawn, P. R.Kemp, P. M. Schofield and D. J. Grainger; Circulation, Vol 90, No. 4,Part 2, p. I-623 (1994); and

“Influence of Human Apo(a) Expression on Fibrinolysis in vivo inTrangenic Mice” T. M. Palabrica, A. C. Liu, M. J. Aronovitz, B. Furie,B. C. Furie and R. Lawn; Circulation, Vol 90, No. 4, Part 2, p. I-623(1994).

On the basis of its suspected thrombogenic activity, Lp(a) has also beenimplicated in peripheral artery disease, in particular stroke. Recentlyclinicians have shown that serum Lp(a) levels were significantly higherin stroke patients than in a reference normal population:

“Lp(a) Lipoprotein in Patients with Acute Stroke” K. Asplund, T. Olsson,M. Viitanen and G. Dahlen; Cerebrovasc. Diseases 1, p. 90-96 (1991).

Restenosis following percutaneous transluminal angioplasty is a commoncomplication occurring in up to 40% of cases within 3-6 months of theintervention. The main cause for restenosis is believed to be abnormalvascular smooth muscle cell activation and proliferation. The proof thathigh plasma Lp(a) levels are associated with smooth muscle cellproliferation and activation was established in vitro and in vivo by thetwo following studies:

“Proliferation of Human Smooth Muscle Cells Promoted by Lipoprotein(a)”D. J. Grainger, H. L. Kirschenlohr, J. C. Metcalfe, P. L. Weissberg, D.P. Wade and R. M. Lawn; Science, Vol 260, p.1655-1658 (1993); and

“Activation of Transforming Growth Factor-β is Inhibited byApolipoprotein (a) in vivo”, D. J. Grainger, P. R. Kemp, A. C. Liu, R.M. Lawn and J. C. Metcalfe; Circulation, Vol 90, No. 4, Part 2, p. I-623(1994).

This observation has led to a hypothesis that associates elevated plasmaLp(a) levels with an increased incidence of restenosis. The hypothesiswas confirmed by the results of a recent clinical study showing that, inpatients with high plasma Lp(a) levels, a reduction of Lp(a) levels bymore than 50% by LDL-apheresis significantly reduced the restenosisrate; see for instance:

“Effectiveness of LDL-Apheresis in Preventing Restenosis AfterPercutaneous Transluminal Coronary Angioplasty (PTCA): LDL-ApheresisAngioplasty Restenosis Trial (L-ART)” H. Yamaguchi, Y. J. Lee, H. Daida,H. Yokoi, H. Miyano, T. Kanoh, S. Ishiwata, K. Kato, H. Nishikawa, F.Takatsu, Y. Kutsumi, H. Mokuno, N. Yamada and A. Noma; Chemistry andPhysics of Lipids, Vol 67/68, p. 399-403(1994).

The above discussion has established the rationale for decreasing plasmaLp(a) in patients at risk with elevated levels (>20-30 mg/dl). The Lp(a)concentration in individuals appears to be highly determined byinheritance and is hardly influenced by dietary regimes. Varioushormones (i.e. steroid hormones, growth hormones, thyroid hormones) havebeen shown to regulate plasma levels of Lp(a) in man. Of particularinterest, drugs which effectively lower LDL such as the bile acidsequestrant cholestyramine or the HMGCoA reductase inhibitors lovastatinor pravastatin do not affect Lp(a) levels. The drugs of the fibratefamily: clofibrate or bezafibrate and the antioxidant drug probucol areequally ineffective. The only drug reported to lower Lp(a) is nicotinicacid. However at the high doses necessary for efficacy (4 g/day)nicotinic acid has several serious side-effects which preclude its wideuse: flushing, vasodilation and hepatotoxicity. Therefore the medicalneed to lower elevated Lp(a) plasma levels, an independent risk factorfor cardiovascular disease, is still unmet.

In contrast to LDL, Lp(a) exists only in mammals high in theevolutionary scale (humans and non human primates) and is exclusivelysynthesized by the liver cells. Cynomolgus monkeys possess Lp(a) that issimilar to human Lp(a), including possession of the uniqueapolipoprotein apo(a). This primate offers an experimental opportunityfor studying the synthesis of Lp(a) and the role of Lp(a) inatherosclerosis and thrombosis. Primary cultures of cynomolgus monkeyhepatocytes have been selected as the in vitro test for screeningaminophosphonate derivatives of formula (I) for their ability tomodulate Lp(a) levels. Prior to screening, this assay system had beenvalidated by testing as reference products nicotinic acid and steroidhormones which are known to lower Lp(a) in man.

The present invention relates to the unexpected discovery thataminophosphonate derivatives are effective for lowering plasma andtissue lipoprotein(a). Accordingly, in a first aspect, the presentinvention provides for the use of a compound of formula (I):

where:

X¹, X², which may be identical or different, are H, a straight orbranched alkyl or alkoxy group having from 1 to 8 carbon atoms, ahydroxy group or a nitro group,

X³ is H, an alkyl group from 1 to 4 carbon atoms, X³O and one of the twoother substituents X¹ or X² may form an alkylidene dioxy ring havingfrom 1 to 4 carbon atoms,

R¹, R², identical or different, are H, a straight or branched alkylgroup having from 1 to 6 carbon atoms,

B is CH₂, CH₂—CH₂ or CH═CH,

n is zero or 1,

Z is H, a straight or branched alkyl group having from 1 to 8 carbonatoms, an acyl group R³—CO where R³ is an alkyl group from 1 to 4 carbonatoms, a perfluoroalkyl group from 1 to 4 carbon atoms,

A is H, CH₂—CH═CH₂, a straight, branched or cyclic alkyl group havingfrom 1 to 8 carbon atoms, or is selected from the following groups:

where k is an integer from 2 to 4, m is 0 or an integer from 1 to 5, X⁴,X⁵, X⁶, identical or different, are H, a straight or branched alkyl oralkoxy group from 1 to 8 carbon atoms, a hydroxy, trifluoromethyl,nitro, amino, dimethylamino, diethylamino group, a halogen atom (F, Cl,Br, I), X⁴ and X⁵ may form an alkylidendioxy ring having from 1 to 4carbon atoms, X⁷ is H or CH₃, R is a straight or branched alkyl grouphaving from 1 to 6 carbon atoms, an aryl or arylalkyl group from 6 to 9carbon atoms;

or a pharmaceutically acceptable salt thereof;

in the manufacture of a medicament for lowering plasma and tissuelipoprotein(a).

European Patent Application EP 0'559'079A (1993) [corresponding to theU.S. Pat. No. 5,424,303] discloses compounds of formula (I) as well astheir use in decreasing plasma cholesterol and blood peroxides.

Preferred compounds of formula (I) for use in the manufacture of amedicament for lowering plasma and tissue lipoprotein(a) are those ofthe formula (Ia):

where B, R¹, R², X¹, X², X³, X⁴, Z, n and m are as hereinbefore defined;

or a pharmaceutically acceptable salt thereof.

Certain compounds within the scope of formula (Ia) are novel and areparticularly useful in lowering plasma and tissue lipoprotein(a).

Accordingly, in a further aspect, this invention providesaminophosphonate derivatives of formula (Ia) where:

X¹ is H, C₍₁₋₈₎alkyl or C₍₁₋₈₎alkoxy;

X² is C₍₁₋₈₎alkyl or C₍₁₋₈₎alkoxy;

X³ is H, C₍₁₋₄₎alkyl, or X³O and one of the two other substituents X¹ orX² may form an alkylidene dioxy ring having from 1 to 4 carbon atoms;

R¹, R², which may be identical or different, are H or C₍₁₋₆₎alkyl;

B is CH₂—CH₂, CH═CH or CH₂;

n is zero or 1;

Z is H or C₍₁₋₈₎alkyl;

m is an integer from 0 to 5;

X⁴ is H, C₍₁₋₈₎alkyl, C₍₁₋₈₎alkoxy, or halo;

and the pyridyl ring is attached by the ring carbon α- or β- to thenitrogen (2- or 3-pyridyl);

or a salt, preferably a pharmaceutically acceptable salt, thereof; andexcluding:

Diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(3-pyridyl)aminomethylphosphonate;

Diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(2-picolyl)aminomethylphosphonate;

Diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(3-picolyl)aminomethylphosphonate;

Diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-methyl-N-(3-picolyl)aminomethylphosphonate;

Diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(2-pyridylethyl)aminomethylphosphonate,and

Diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(4-picolyl)aminomethylphosphonate.

Suitably, X¹ is H, C₍₁₋₄₎alkyl or C₍₁₋₄₎alkoxy, preferably C₍₁₋₃₎alkylor C₍₁₋₃₎alkoxy, more preferably hydrogen, methyl or methoxy.

Suitably, X² is C₍₁₋₄₎alkyl or C₍₁₋₄₎alkoxy, preferably C₍₁₋₃₎alkyl orC₍₁₋₃₎alkoxy, more preferably methyl or methoxy.

Suitably, X¹ and X² are both alkoxy or one of X¹ and X² is alkyl and theother is alkoxy, or one of X¹ and X² is C₍₁₋₄₎alkyl and the other of X¹and X² is C₍₁₋₃₎alkyl.

Suitable combinations of X¹ and X² include methoxy and methoxy, methoxyand methyl, n-propyl or isobutyl, methyl and methyl or t-butyl,respectively.

Preferably, X³ is hydrogen.

Preferably, (B)_(n) is a direct bond.

Preferably, R¹ and R² is each a C₍₁₋₃₎alkyl group, more preferably, a C₂or C₃ alkyl group, in particular R¹ and R² is ethyl or isopropyl.

Preferably, Z is hydrogen.

Preferably, X⁴ is hydrogen or methyl which is preferably on the ringcarbon adjacent to N.

Preferably, the pyridyl ring is attached by the ring carbon β- to thenitrogen (3-pyridyl).

When used herein, the terms ‘alkyl’ and ‘alkoxy’ include both straightand branched groups, for instance, methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, s-butyl, t-butyl, etc.

Preferred compounds of formula (Ia) include:

Diisopropylα-(4-hydroxy-3-methoxy-5-methylphenyl)-N-(3-pyridyl)-aminomethylphosphonate;

Diisopropylα-(3,5-dimethoxy-4-hydroxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate;

Diethylα-(3-methyl-4-hydroxy-5-t-butylphenyl)-N-(3-pyridyl)-aminomethylphosphonate;

Diethylα-(3,5-dimethoxy-4-hydroxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate;and

Diethylα-(3,5dimethyl-4-hydroxyphenyl)-N-(3pyridyl)-aminomethylphosphonate.

Independently from the previously published activity, the presentinvention relates to the unexpected discovery that aminophosphonatederivatives of formula (I) are effective for decreasing Lp(a) productionby primary cultures of Cynomolgus monkey hepatocytes. Lp(a) of theseprimates is similar in immunologic properties to human Lp(a) and occursin an almost identical frequency distribution of plasma concentrations,see for instance:

“Plasma Lipoprotein(a) Concentration is Controlled by Apolipoprotein(a)Protein Size and the Abundance of Hepatic Apo(a) mRNA in a CynomolgusMonkey Model”, N. Azrolan, D. Gavish and J. Breslow; J. Biol. Chem., Vol266, p. 13866-13872 (1991).

Therefore the compounds of this invention are potentially useful fordecreasing Lp(a) in man and thus provide a therapeutic benefit.

In particular, this invention provides a new therapeutic use foraminophosphonate compounds of formula (I) as Lp(a) lowering agents.Diseases associated with elevated plasma and tissue levels oflipoprotein(a) include, for instance, coronary heart disease, peripheralartery disease, intermittent claudication, thrombosis, restenosis afterangioplasty, extracranial carotid atherosclerosis, stroke andatherosclerosis occuring after heart transplant.

The recently discovered Lp(a) lowering activity of the aminophosphonatesof formula (I) is independent from their previously reportedpharmacological activities of decreasing plasma cholesterol and bloodperoxides. Recent clinical studies have shown that neither thehypocholesterolemic drug pravastatin nor the antioxidant drug probucolcan decrease Lp(a) levels in man. See for example:

“Serum Lp(a) Concentrations are Unaffected by Treatment with the HMG-CoAReductase Inhibitor Pravastatin: Results of a 2-Year Investigation” H.G. Fieseler, V. W. Armstrong, E. Wieland, J. Thiery, E. Schiltz, A. K.Walli and D. Seidel; Clinica Chimica Acta, Vol 204, p. 291-300 (1991);and

“Lack of Effect of Probucol on Serum Lipoprotein(a) Levels”, A. Noma;Atherosclerosis 79, p. 267-269 (1989).

For therapeutic use the compounds of the present invention willgenerally be administered in a standard pharmaceutical compositionobtained by admixture with a pharmaceutical carrier selected with regardto the intended route of administration and standard pharmaceuticalpractice. For example, they may be administered orally in the form oftablets containing such excipients as starch or lactose, or in capsule,ovules or lozenges either alone or in admixture with excipients, or inthe form of elixirs or suspensions containing flavouring or colouringagents. They may be injected parenterally, for example, intravenously,intramuscularly or subcutaneously. For parenteral administration, theyare best used in the form of a sterile aqueous solution which maycontain other substances, for example, enough salts or glucose to makethe solution isotonic with blood. The choice of form for administrationas well as effective dosages will vary depending, inter alia, on thecondition being treated. The choice of mode administration and dosage iswithin the skill of the art.

The compounds of structure (I) and their pharmaceutically acceptablesalts which are active when given orally can be formulated as liquids,for example syrups, suspensions or emulsions or as solids for example,tablets, capsules and lozenges. A liquid formulation will generallyconsist of a suspension or solution of the compound or pharmaceuticallyacceptable salt in a suitable liquid carrier(s) for example, ethanol,glycerine, non-aqueous solvent, for example polyethylene glycol, oils,or water with a suspending agent, preservative, flavouring or colouringagents.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidformulations. Examples of such carriers include magnesium stearate,starch, lactose, sucrose and cellulose.

A composition in the form of a capsule can be prepared using routineencapsulation procedures. For example, pellets containing the activeingredient can be prepared using standard carriers and then filled intoa hard gelatin capsule; alternatively, a dispersion or suspension can beprepared using any suitable pharmaceutical carrier(s), for exampleaqueous gums, celluloses, silicates or oils and the dispersion orsuspension then filled into a soft gelatin capsule.

Typical parenteral compositions consist of a solution or suspension ofthe compound or pharmaceutically acceptable salt in a sterile aqueouscarrier or parenterally acceptable oil, for example polyethylene glycol,polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.Alternatively, the solution can be lyophilised and then reconstitutedwith a suitable solvent just prior to administration.

A typical suppository formulation comprises a compound of structure (I)or a pharmaceutically acceptable salt thereof which is active whenadministered in this way, with a binding and/or lubricating agent suchas polymeric glycols, gelatins or cocoa butter or other low meltingvegetable or synthetic waxes or fats.

Preferably the composition is in unit dose form such as a tablet orcapsule.

Each dosage unit for oral administration contains preferably from 1 to250 mg (and for parenteral administration contains preferably from 0.1to 25 mg) of a compound of the structure (I) or a pharmaceuticallyacceptable salt thereof calculated as the free base.

The pharmaceutically acceptable compounds of the invention will normallybe administered to a subject in a daily dosage regimen. For an adultpatient this may be, for example, an oral dose of between 1 mg and 500mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous,or intramuscular dose of between 0.1 mg and 100 mg, preferably between0.1 mg and 25 mg, of the compound of the structure (I) or apharmaceutically acceptable salt thereof calculated as the free base,the compound being administered 1 to 4 times per day.

Compounds of formula (I) may be prepared according to the processesdescribed in European Patent Application EP 0 559 079-A(1993[corresponding to the U.S. Pat. No. 5,424,303]. This process whichhas two variants is shown in the following general scheme:

Variant 1 is used when Z is H, i.e. when the starting compound is aprimary amine. Briefly, the aminophosphonates of formula (I) areprepared by nucleophilic addition of a dialkyl phosphite or its sodiumsalt obtained in situ by the reaction of dialkyl phosphite and sodiumhydride on the imine obtained by condensation of the appropriatealdehyde and a primary amine.

Variant 2 is used when Z is not H, i.e. when the starting compound is asecondary amine. In this case, the aminophosphonates of formula (I) areprepared by reacting equimolar amounts of the appropriate aldehyde andthe secondary amine and a dialkyl phosphite. The reaction isadvantageously carried out in the presence of p-toluenesulfonic acid asa catalyst in a hydrocarbon solvent such as benzene or toluene withconcomittant elimination of water, for instance, by using a Dean-Starkapparatus.

Novel compounds of formula (Ia) in which Z is hydrogen may be preparedby a process which comprises treating an imine of formula (II):

in which B, X¹, X², X³, X⁴, m and n are as hereinbefore defined;

with a phosphite compound of formula (III):

HPO(OR¹)(OR²)  (III)

in which R¹ and R² are as hereinbefore defined; or a trialkyl silylderivative thereof, preferably the trimethyl silyl phosphite, or a metalsalt thereof, for instance the sodium salt, formed in situ by treatmentof the compound of formula (III) with a suitable base, for instancesodium hydride, ethoxide or methoxide.

The reaction may be carried out in the presence or absence of acatalyst. Suitable catalysts include amine such as diethylamine ortriethylamine. The reaction may be carried out in the absence orpresence of a solvent. Suitable solvents include petroleum ether,benzene, toluene, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane.Suitable reaction temperatures are in the range 30 to 140° C.

The imine compound of formula (II) may be obtained by condensing analdehyde compound of formula (V):

in which B, X¹, X², X³ and n are as hereinbefore defined;

with a primary amine of formula (VI):

H₂NA  (VI)

in which A is as hereinbefore defined;

under imine forming conditions.

Suitably, the condensation may be effected with or without a catalyst ina solvent such as ether, tetrahydrofuran, benzene, toluene or ethanol.Suitable catalysts include molecular sieve, an acid such as glacialacetic acid, p-toluene sulphonic acid, thionyl chloride, titaniumtetrachloride, boron trifluoride etherate, or a base such as potassiumcarbonate. The reaction is suitably carried out at a temperature in therange 0° C. to the boiling point of the solvent being used. For lessreactive amines/aldehydes, the reaction may be usefully carried out in aDean-Stark apparatus.

Novel compounds of formula (Ia) in which Z is not hydrogen may beprepared by a process which comprises treating equimolar amounts of analdehyde of formula (V), a secondary amine of formula (VII):

HNZA  (VII)

in which Z is a C₍₁₋₈₎alkyl group and A is as hereinbefore defined; and

a phosphite of formula (III), suitably in the presence ofp-toluenesulfonic acid as a catalyst, in a hydrocarbon solvent such aspetroleum ether, benzene, toluene or xylene, at a temperature betweenambient temperature and the boiling point of the solvent being used, andwith concomittant elimination of water, for instance, by using aDean-Stark apparatus.

Compounds of formula (Ia) in which m is not zero may also be prepared bya process which comprises treating a compound of formula (VIII):

in which B, R¹, R², X¹, X², X³ and n are as hereinbefore defined;

an aldehyde of formula (IX):

in which m is an integer from 1 to 5 and X⁴ is as hereinbefore defined;

under reductive amination conditions.

Suitable such conditions include carrying out the reaction in thepresence of sodium cyanoborohydride in an alcoholic solvent, preferablymethanol, at a pH between 3 to 6 and at a temperature between 0° C. and25° C.

A compound of formula (VIII) may be obtained according to the processhereinbefore described for a compound of formula (Ia) from an aldehydeof formula (V), a secondary amine of formula (VII) in which Z isprotecting group which can be removed by hydrogenolysis, for instance anα substituted benzyl or bezyloxycarbonyl and a phosphite of formula(III). This forms an intermediate which is then subjected tohydrogenolysis according to standard conditions, to give a compound offormula (VIII).

Through their amino function, the aminophosphonate ester (I) can formsalts of inorganic acids such as HCl, H₂SO₄ or with organic acids suchas oxalic acid, maleic acid, sulfonic acids, etc. An example ofhydrochloride salt of aminophosphonate (I) is provided (example 5). Allthese salts are integral part of this invention.

Compounds of structure (I) are racemates as they have at least onechiral center which is the carbon atom in position alpha to thephosphonate group. The compounds (I) therefore exist in the twoenantiomeric forms. The racemic mixtures (50% of each enantiomer) andthe pure enantiomers are comprised in the scope of this application. Incertain cases, it may be desirable to separate the enantiomers.

In a further aspect, the present invention provides a process for theenantiomeric synthesis of a derivative of formula (I) which processcomprises treating either of the (+) or (−) enantiomer of theα-substituted aminomethylphosphonate of formula (X):

in which B, R¹, R², X¹, X², X³ and n are as hereinbefore defined;

with an aldehyde of formula (XI):

R³—CHO  (XI)

in which R³ is as hereinbefore defined;

under reductive amination conditions.

Suitable such conditions include carrying out the reaction in thepresence of sodium cyanoborohydride in an alcoholic solvent, preferablymethanol, at a pH between 3 to 6 and at a temperature between 0° C. and25° C.

The key α-substituted primary aminomethylphosphonate of formula (X) isobtained by treating an aldehyde of formula (V), as hereinbeforedefined, with (+) or (−)α-methylbenzylamine to form an intermediateimine which is then reacted with a phosphite ester HPO(OR¹)(OR²) to givea mixture of diastereoisomers which may be separated by conventionaltechniques, for instance fractional crystallisation or chromatography.Hydrogenolysis can then be used to remove the benzyl group fromnitrogen, to give the α-substituted primary aminomethyl-phosphonate offormula (X). This approach is illustrated by the preparation ofenantiomers of compounds No. 7 and 15 of Table 1. Alternately. theresolution of the aminophosphonate racemates can be effected bypreparative chiral chromatography, in particular chiral HPLC. Theexperimental conditions for chromatographic separation of enantiomers ofcompound No. 20 are provided. With either separation method, finalenantiomeric purity can be ascertained by measuring the specificrotations of the separated isomers.

The structure of compounds of formula (I) were established by theirelemental analysis, their infrared (IR), mass (MS) and nuclear magneticresonance (NMR) spectra The purity of the compounds was checked by thinlayer, gas liquid or high performance liquid chromatographies.

The invention is further described in the following examples which areintended to illustrate the invention without limiting its scope. In thetables, n is normal, i is iso, s is secondary and t is tertiary. In thedescription of the NMR spectra, respectively s is singlet, d doublet, ttriplet and m multiplet TsOH is p-toluenesulfonic acid monohydrate. Thetemperatures were recorded in degrees Celsius and the melting points arenot corrected. In the measurement of optical activity, an enantiomerwhich rotates the plane of polarized light to the right is calleddextrorotatory and is designated (+) or (D). Conversely, levorotatorydefines an enantiomer which rotates the plane of polarized light to theleft, designated (−) or (L). Unless otherwise indicated, the physicalconstants and biological data given for aminophosphonates of formula (I)refer to racemates.

EXAMPLE 1 Dimethylα-(3,5-di-tert-Butyl-4-hydroxyphenyl)-N-(3-pyridyl)-amino-methylphosphonate

A mixture of 50 g (0.206 mol) of 3,5-di-tert-butyl-4-hydroxybenzaldehydeand 20.3 g (2.16 mol) of 3-aminopyridine dissolved in 300 ml toluene anda catalytic amount of p-toluenesulfonic acid (ca. 50 mg) contained in aflask, connected to a Dean Stark apparatus was refluxed for 17 h. Thesolution was evaporated to dryness to give a solid which was purified byrecrystallisation from ligroin: mp=125-130°, IR (KBr): 1590 cm⁻¹: CH═N.

Dimethyl phosphite (63.8 g, 0.58 mol) was added to 60 g (0.19 mol) ofthe previously described imine dissolved in 230 ml THF and the mixturewas refluxed for 6 h. The solvent was evaporated and the residue waspurified by column chromatography (SiO₂, 9/1 CHCl₃/MeOH).Recrystallisation from a mixture of methyl-tert-butyl ether/petroleumether gave a white solid, mp=168-170° C.

IR (KBr)=3300 cm⁻¹: NH, 1240: P═O, 1030: P—O—C; NMR (CDCl₃): δ=8.06,7.96, 7.4 and 6.9 (4m, 1H each): aromatic H, 3-pyridyl, 7.2 (d,J_(P—H)=2 Hz, 2H): aromatic H, substituted phenyl, 5.24 (s, 1H): OH,4.66 (d, J_(P—H)=22 Hz, 1H): CH—PO₃Me₂, 4.75-4.68 (m, 1H): NH, 3.74 and3.39=(two d, J=11 Hz): P—O—CH ₃, 1.42 (s, 18H): tert-Bu; MS: m/e=419:M⁺−1,311 (100%): M⁺ —PO₃Me₂.

EXAMPLE 2 Diethylα-(3,5-di-tert-Butyl-4-hydroxyphenyl)-N-(2-pyridyl)-amino-methylphosphonate

The process described in example 1 was employed using 2-aminopyridine asthe amine and diethyl phosphite as the phosphonate reagent. The titlecompound was purified by column chromatography (95/5 CHCl₃/MeOH) toyield a solid (61%); mp=116-118° (AcOEt-ligroin).

MS (m/e)=448: M⁺, 311: M⁺ —PO₃Et₂, 78 (100%): C₅H₄N; δ=8.09, 7.38 and6.57 and 6.44 (4m, 1H each): aromatic H, 2-pyridyl, 7.28 (d, J_(P—H)=2Hz, 2H): aromatic H, substituted phenyl, 5.46 (dd, J=9 and 22 Hz, 1H):CH—PO₃Et₂, 5.3 (m, 1H): N—H, 4.14-3.66 (3m, 4H total): P—O—CH ₂—CH3,1.42 (s, 18H): tert-Bu, 1.21 and 1.16 (2 t, 3H each): P—O—CH₂—CH ₃.

EXAMPLE 3 Diethylα-(3,5-di-tert-Butyl-4-hydroxyphenyl)-N-[5-(2-chloropyridyl)]-aminomethylphosphonate

The process described in example 1 was employed using5-amino-2-chloropyridine as the amine and diethyl phosphite as thephosphonate reagent. The title compound was obtained in 50% yield aftercolumn chromatography (98/2 CHCl₃/MeOH) and trituration in petroleumether, mp=124-126° C.

MS (m/e)=483: M⁺+1,345 (100%), 347 (30%): M⁺—PO₃Et₂: NMR (CDCl₃):δ=7.78, 7.05 and 6.09 (3H): aromatic H, 3-pyridyl, 7.18 (d, J=2 Hz, 2H):aromatic H, substituted phenyl, 5.22 (s, 1H): OH, 4.83 (t, J=8 Hz): N—H,4.57 (dd, J=7.5 and 22.5 Hz): CH—PO₃—Et₂, 4.1, 3.86 and 3.56 (3m, 4H):P—O—CH ₂—CH₃, 1.40 (s, 18H): t-Bu, 1.28 and 1.05 (2t, J=7 Hz):P—O—CH₂—CH ₃.

EXAMPLE 4 Diethylα-(3,5-di-tert-Butyl-4-hydroxyphenyl)-N-acetyl-N-(4-picolyl)-aminomethylphosphonate

A mixture of acetic anhydride (1.4 g, 14 mmol), diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(4-picolyl)-aminomethylphosphonate(6 g, 13 mmol) and triethyl amine (1.9 ml, 14 mmol) in 20 ml toluene wasrefluxed for 16 h. The reaction mixture was extracted with brine, driedand evaporated to dryness. The residue was recrystallized in a mixtureof dichloromethane and petroleum ether to give 3.7 g (57% yield);mp=160-162° C.

MS (m/e): 504: M⁺, 461: M⁺ —COCH₃, 367: M⁺ —PO₃—Et₂, 325 (100%): M⁺+1-PO₃Et₂—COCH₃.

EXAMPLE 5 Hydrochloride Salt of Diethylα-(3,5-di-tert-Butyl-4-hydroxyphenyl)-N-(3-pyridyl)aminomethylphosphonate

Diethyl α-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(3-pyridyl)aminomethylphosphonate (3 g, 6.7 mmol) was dissolved with slight warmingin 60 ml toluene and the resulting solution was saturated with gaseoushydrogen chloride. After 16 h at 0° C. the mixture was evaporated todryness and the residue was recrystallized in EtOH; mp=193-194° C.

% Calc. C 59.43 H 7.90 Cl 7.31 N 5.78 P 6.39 % Found C 59.53 H 8.10 Cl7.02 N 5.72 P 6.21

EXAMPLE 6 Diethylα-(3,4-Methylenedioxy-N-(3-pyridyl)-aminomethylphosphonate

The process described in example 8 was followed. The title compound waspurified by column chromatography (9/1 CHCl₃/MeOH); 60% yield, mp=98-99°C., C₁₇H₂₁N₂O₅P.

IR (KBr)=1240 cm⁻¹: P═O, 1030: P—O—C; MS (m/e)=365 M⁺ +1, 227 (100%): M⁺—PO₃Et₂; NMR (CDCl₃) δ=8.1, 7.95, 7.05 and 6.95 (4m, 1H each): aromaticH, 3-pyridyl, 6.90, 6.85, 6.75 (3m, 3H): aromatic H, substituted phenyl,5.95 (2H): ═O—CH ₂—O, 4.86 (d×d, 1H, J=8 and 10 Hz): N—H, 4.63 (d×d, 1H,J=8 and 24 Hz): CH—PO₃Et₂, 4.18-3.70 (3m, 4H total): P—O—CH ₂—CH₃, 1.31and 1.16: (2t, J=7 Hz): P—O—CH₂—CH ₃.

EXAMPLE 7 Diethylα-(4-Hydroxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate

4-Hydroxybenzaldehyde (6 g, 49 mmol) was reacted at room temperaturewith 3-aminopyridine (4.5 g, 52 mmol) in 30 ml THF at room temperatureto give 9.9 g of a light brown solid. The imine so obtained (5.9 g, 30mmol) was dissolved in 50 ml THF, diethyl phosphite was added in twoportions, one at the beginning of the reaction and the other after 6 hat reflux, (total amount: 8.2 g, 60 mmol). The reaction mixture wasrefluxed overnight. Filtration of the precipitate formed gave 7.5 g(75%) of a tan solid, mp=210-212° C. (EtOH).

MS (m/e)=337: M⁺ +1, 199 (100%): M⁺ —PO₃Et₂; NMR (DMSO-d6): δ=9.35 (s,1H); OH, 8.15, 7.7, 7.1 and 7.0 (4m, 1H each): aromatic H, 3-pyridyl,6.5 (d×d, 1H): N—H, 7.3 and 6.7 (2m, 2H each): aromatic H,4-hydroxyphenyl, 4.93 (d×d, 1H): CH—PO₃Et₂, 4.1-3.6 (3m, 4H total):P—O—CH ₂—CH₃, 1.15 and 1.02 (2t, 3H each): P—O—CH₂—CH ₃.

EXAMPLE 8 Diethylα-(3,5-Dimethoxy-4-hydroxyphenyl)-N-(3-pyridyl)amino-methylphosphonate

A mixture of 3 g (16.4 mmol) of syringaldehyde and 1.63 g (17.3 mmol) of3-aminopyridine dissolved in 10 ml toluene and a catalytic amount ofp-toluenesulfonic acid (ca. 5 mg) contained in a flask connected to aDean Stark apparatus was refluxed for 17 h. The solution was evaporatedto dryness to give 4.2 g (100%) of the crude imine. Diethyl phosphite(4.8 g, 35 mmol) was added to 4.2 g (17.3 mmol) of the previouslydescribed imine dissolved in 10 ml THF and the mixture was refluxed for7 h. Another amount of diethyl phosphite (4.8 g, 35 mmol) was added andthe mixture was refluxed overnight (total reaction time: 17 h). Thesolvent and the excess of diethyl phosphite were evaporated and theresidue was recrystallized from a mixture of ethanol and dichloromethaneto give 4.2 g (61%) of a white solid, mp=181-183°.

IR (KBr)=1240 cm⁻¹ : P═O and 1030: P—O—C; MS (m/e)=397: M⁺ +1,259(100%): M⁺ —PO₃Et₂; NMR (CDCl₃): δ=8.08, 7.98, 7.04 and 6.84 (4m, 1Heach): aromatic H, 3-pyridyl, 6.69 (d, J=2 Hz, 2H): aromatic H,substituted phenyl, 5.8 (broad, 1H): OH, 4.84 (d×d, 1H, J=7 and 10 Hz):N—H, 4.62 (d×d, 1H, J=7 and 23 Hz): CH—PO₃Et₂, 4.18-3.65 (3m, 4H total):P—O—CH ₂—CH₃, 3.86 (s, 6H): OCH ₃, 1.31 and 1.16: (2t, J=7 Hz):P—O—CH₂—CH ₃.

Elemental analysis: C₁₈H₂₅N₂O₆P % Calc. C 54.54 H 6.36 N 7.07 P 7.81 %Found C 54.50 H 6.38 N 6.99 P 7.65

EXAMPLE 9 Diethylα-(3,4,5-Trimethoxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate

A mixture of 3,4,5-trimethoxybenzaldehyde (10 g, 51 mmol) and3-aminopyridine (4.8 g, 51 mmol) and a catalytic amount of TsOH in 50 mltoluene was refluxed for 16 h in a flask connected to a Dean-Stark trap.Evaporation of toluene gave 12.9 g (93%) of the crude imine which wasused directly in the next reaction. A 50 ml THF mixture containing theimine (6g, 22 mmol) and diethyl phosphite (6.1 g introduced at thebeginning and 6.1 g after 4 h, total amount=12.2 g, 88 mmol) wasrefluxed for 8 h. The residue after evaporation of THF and excess ofHPO₃Et₂ was triturated in petroleum ether to give 7.12 g (79%) of awhite solid, mp=135-137° C.

MS (m/e)=410: M⁺, 273 (100%): M⁺ —PO₃Et₂; NMR (CDCl₃): δ=8.1, 8.0, 7.05and 6.85 (4m, 1H each): aromatic H, 3-pyridyl, 6.69 and 6.68: (d, J=2Hz, 2H): aromatic H, substituted phenyl, 4.86 (d×d, 1H, J=8 and 10 Hz):N—H, 4.63 (d×d, 1H, J=7 and 23 Hz): CH—PO₃Et₂, 4.18-3.70 (3m, 4H total):P—O—CH ₂—CH₃, 3.86 (two s, 9H): OCH ₃, 1.31 and 1.16: (2t, J=7 Hz):P—O—CH₂—CH ₃.

EXAMPLE 10 Diethylα-(3-Ethoxy-4-hydroxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate

A 50 ml toluene solution containing 3-ethoxy-4-hydroxybenzaldehyde (10g, 60 mmol), 3-aminopyridine (5.6 g, 60 mmol) and 50 mg of TsOH placedin a flask connected to a Dean-Stark trap was refluxed for 4 h to give14.62 g (95%) of the corresponding imine.

To a suspension of sodium hydride (1.19 g of a 60% mixture, 30 mmol) in20 ml dry THF was added HPO₃Et₂ (9.12 g 66 mmol) under nitrogen and theresulting mixture was stirred until the initial turbid suspension becamecompletely clear. To this solution of NaPO₃Et₂ was added the above imine(8 g, 33 mmol) dissolved in 10 ml THF and the resulting solution wasrefluxed for 2 h. THF was evaporated and the residue was partitionedinto H₂O and CH₂Cl₂. Evaporation of the dried organic phase gave 3.1 gof a white solid, mp=184-187° C.

MS: (m/e)=380: M⁺, 243: M⁺ —PO₃Et₂; NMR (DMSO-d6): δ=8.9 (s, 1H): OH,8.15, 7.3, 7.0 and 6.9 (1H each): aromatic H, 3-pyridyl, 7.1 (m, 2H) and6.68 (d, J=8 Hz, 1H): aromatic H, phenyl, 6.5 (d×d, J=6 and 10 Hz): NH,4.92 (d×d, J=10 and 24 Hz): CH—PO₃Et₂, 4.05-3.6 (4m, 6H total): P—O—CH₂—CH₃ and OCH ₂ CH₃, 1.29 (t, J=7 Hz, 3H): O—CH₂—CH ₃, 1.16 and 1.04(2t, J=7 Hz, 3H each): P—O—CH₂—CH ₃.

EXAMPLE 11 Diethylα-(4-Hydroxy-3-methoxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate

The procedure described in example 10 was followed, using4-hydroxy-3-methoxybenzaldehyde as the starting material. The titlecompound is a white solid, mp=170-173° C.

MS (m/e)=366: M⁺, 229: M⁺ —PO₃Et₂; NMR (DMSO-d6) δ=8.9 (s, 1H): OH,8.15, 7.75, 7.0 and 6.9 (4m, 4H): aromatic H, 3-pyridyl, 7.1 (m, 2H) and6.7 (d, J=8 Hz, 1H): aromatic H, phenyl, 6.5 (d×d, J=6 and 10 Hz): NH,4.92 (d×d, J=10 and 24 Hz): CH—PO₃Et₂, 4.05-3.6 (3m, 4 H total): P—O—CH₂—CH₃, 3.72 (s, 3H): OCH₃, 1.17 and 1.4 (2t, J=7 Hz, 6H): P—O—CH₃—CH ₃.

EXAMPLE 12 Diethylα-(3,5-Dimethoxy-4hydroxyphenyl)-N-(4-picolyl)-aminomethylphosphonate

A solution of 2.5 g (13.7 mmol) syringaldehyde and 1.6 g (14.4 mmol)4-picolylamine dissolved in 100 ml toluene contained in a flaskconnected to a Dean-Stark apparatus was refluxed for 3 h. Toluene wasevaporated under vacuum then the residue dissolved in 10 ml THF washeated with 5.1 g (36.8 mmol) diethyl phosphite for 6 h. THF wasevaporated and the residue was purified by column chromatography (SiO₂,95/5 CHCl₃/MeOH). Recrystallisation in a mixture of CH₂Cl₂-petroleumether gave 3.7 g (45%) of a solid, mp=124-126° C.

MS (m/e)=410: M⁺, 273: M⁺ —PO₃Et₂; NMR (CDCl₃) δ=8.55 and 7.22 (2m, 4H):aromatic H, 4-picolyl, 6.75 (d, J=2 Hz, 2H): aromatic H, phenyl,4.15-3.77 (several m, 5 H): P—O—CH ₂—CH₃ and CH—PO₃Et₂, 3.89 (s, 6H):OCH₃, 3.82 and 3.62 (2d, J=14 Hz): NH—CH ₂—Py, 1.33 and 1.16 (2t, J=7Hz, 6H): P—O—CH₂—CH ₃.

EXAMPLE 13 Diethylα-(3,5-Dimethoxy-4-hydroxyphenyl)-N-(3-picolyl)-aminomethylphosphonate

The procedure described in example 12 was followed, using 3-picolylamineas the starting material. The title compound was purified by columnchromatography (9/1 CHCl₃/MeOH) to give a thick yellow oil.Recrystallization from CH₂Cl₂-Petroleum ether gave a tan solid,mp=99-101°.

MS (m/e): 410: M⁺, 273 =M⁺ —PO₃Et2; NMR (CDCl₃) δ=8.51, 8.50, 7.64 and7.25 (4m, 4H): aromatic H, 3-picolyl, 6.65 (d, J=2 Hz, 2H): aromatic H,phenyl, 7.75 (broad, 1H): OH, 4.15-3.75 (several m, 5H): P—O—CH ₂—CH₃and CH—PO₃Et₂, 3.9 (s, 6H): OCH₃, 3.82 and 3.61 (2d, J=14 Hz, 2H): NH—CH₂—Py, 1.31 and 1.16 (2t, J=7 Hz, 6H): P—O—CH₂—CH ₃.

EXAMPLE 14 Diethylα-(3,5-Dimethoxy-4-hydroxyphenyl)-N-(2-pyridyl)-amino-methylphosphonate

A mixture of 3.64 g (20 mmol) of syringaldehyde and 1.88 g (20 mmol) of2-aminopyridine dissolved in 20 ml toluene and a catalytic amount ofTsOH contained in a flask connected to a Dean Stark apparatus wasrefluxed for 24 h. The solution was evaporated to dryness to give 5.2 g(100%) of the crude imine. Diethyl phosphite (5.8 g, 42 mmol) was addedto 3.6 g (14 mmol) of the previously described imine dissolved in 25 mlTHF and the mixture was refluxed for 20 h. The solvent and the excess ofdiethyl phosphite were evaporated and the residue was recrystallizedfrom ethanol to give 4.2 g (76%) of a white solid. mp=163-165° C.

IR (KBr)=1240 cm⁻¹: P═O and 1030: P—O—C; MS (m/e)=397: M⁺ +1, 259(100%): M⁺ —PO₃Et₂; NMR (CDCl₃): δ=8.08, 7.37, 6.60 and 6.41 (4m, 1Heach): aromatic H, 2-pyridyl, 6.76 (d, J=2 Hz, 2H): aromatic H,substituted phenyl, 5.6 (s, 1H): OH, 5.39 (m, 1H): N—H, 5.37 (d×d, 1H,J=9 and 28 Hz): CH—PO₃Et₂, 4.18-3.69 (3m, 4H total): P—O—CH ₂—CH₃, 3.87(s, 6H): OCH ₃, 1.24 and 1.15: (2t, J=7 Hz): P—O—CH₂—CH ₃.

EXAMPLE 15 Diethylα-(3,5-Dimethoxy-4-hydroxyphenyl)-N-(4-pyridyl)-aminomethyl Phosphonate

A 25 ml toluene solution containing syringaldehyde (3.64 g, 20 mmol), 4aminopyridine (1.9 g, 20 mmol) and 5 mg of TsOH placed in a flaskconnected to a Dean-Stark trap was refluxed for 48 h to give 5.0 g (95%)of the corresponding imine.

To a suspension of sodium hydride (0.87 g of a 60% mixture, 20 mmol) in25 ml dry THF was added HPO₃Et₂ (4.14 g, 30 mmol) under nitrogen and theresulting mixture was stirred until the initial turbid suspension becamecompletely clear. To this solution of NaPO₃Et₂ was added the above imine(2.6 g, 10 mmol) dissolved in 5 ml THF and the resulting solution wasrefluxed for 2 h. THF was evaporated and the residue was partitionedinto H₂O and CH₂Cl₂. Evaporation of the dried organic phase gave a whitesolid which was recrystallized in EtOH (1.84 g, 45%); mp=172-174° C.

MS (m/e)=396: M⁺, 259 (100%): M⁺ —PO₃Et₂; NMR (CDCl₃): δ=8.18, 8.16,6.48 and 6.46 (4m, 1H each): aromatic H, 4-pyridyl, 6.67 (d, J=2 Hz,2H): aromatic H, substituted phenyl, 5.27 (d×d, 1H, J=7 and 10 Hz): N—H,4.66 (d×d, 1H, J=7 and 23 Hz): CH—PO₃Et₂, 4.18-3.60 (3m, 4H total):P—O—CH ₂—CH₃, 3.87 (s, 6H): OCH ₃, 1.30 and 1.15: (2t, J=7 Hz):P—O—CH₂—CH ₃.

EXAMPLE 16 Enantiomers of Diethylα-(3,5-di-tert-Butyl-4-hydroxyphenyl)-N-(4-picoyl)-aminomethylphosphonate

a) 3,5-Di-tert-butyl-4-hydroxybenzaldehyde (30 g, 123.5 mmol) and(R)-(+)-1-phenyl-ethylamine (15.7 g, 129.7 mmol) were stirred in 100 mlof THF at room temperature for one day. The solution was dried on MgSO₄and concentrated. The corresponding imine was recrystallized fromligroin (38 g; 88% yield; mp=127-128° C.).

The imine (30 g, 89 mmol) and diethylphosphite (15.4 g, 111.3 mmol) wererefluxed in 80 ml of toluene for 5 hours. The mixture was evaporated todryness. HPLC assay of the residue showed that one of the diastereomersis formed predominantly (84% vs 3% of the reaction mixture). The majordiastereomer of diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(1-phenyl-ethyl)-aminomethylphosphonatewas isolated by successive crystallizations (10 g); [α]_(D) ²⁷+8.33°(c=1.649, CHCl₃); mp=105-106° C.). (+)-Diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(1-phenyl-ethyl)-aminomethylphosphonate(9.5 g, 20 mmol) was hydrogenated in ethanol in the presence of 2.5 g of10% Pd on charcoal to give (−)-diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-aminomethylphosphonate (5.6 g; 76%yield; mp=143-145° C. (recrystalized from ligroin/CH₂Cl₂); [α]_(D)²²−12.12° (c=1.650, CHCl₃).

(−)-Diethyl α-(3,5-di-tert-butyl-4-hydroxyphenyl)-aminomethylphosphonate(11 g, 29.6 mmol) and pyridine 4carboxaldehyde (6.3 g, 59.3 mmol) weredissolved in 125 ml of MeOH. The mixture was acidified with concentratedHCl (blue bromophenol indicator). After half an hour of stirring at roomtemperature, NaBH₃CN (5.6 g, 89 mmol) dissolved in 30 ml MeOH was addedand the pH was adjusted again with HCl. The reaction mixture was stirredat room temperature for 4 hours then evaporated to dryness and extractedwith CH₂Cl₂ and water. The organic phase was dried over MgSO₄ andevaporated. The residue was separated by column chromatography(silicagel, 95/5 CHCl₃/MeOH to give (−)-diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(4-picolyl)-aminomethylphosphonate[(11 g; 80% yield; mp=66-69° C.; [α]_(D) ²¹−44.05° (c=1.992, CHCl₃)].

b) 3,5-Di-tert-butyl-4-hydroxybenzaldehyde (30 g, 123.5 mmol) and(S)-(−)-1-phenyl-ethylamine (15.7 g, 129.7 mmol) were stirred in 100 mlof THF for one day to give the corresponding imine (36.5 g; 88% yield;mp=127-128° C.).

The imine (20 g, 59.3 mmol) and diethylphosphite (10.2 g, 74.2 mmol)were refluxed in 60 ml of toluene for 7 hours. The mixture wasevaporated to dryness. HPLC assay of the residue indicated thediastereomeric ratio to be 60 to 40% in addition to starting materials.The latter were stripped off by column chromatography on silicagel (98/2CH₂Cl₂/MeOH). The fractions containing the mixture of diastereomers wereevaporated to dryness and recrystallized three times from ligroin/MTBEto yield the major diastereomer of diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(1-phenyl-ethyl)-aminomethylphosphonate)[12 g; mp=104-105° C.; [α]_(D) ²⁸−10.53° (c=1.643, CHCl₃)].

(−)-Diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(1-phenyl-ethyl)-aminomethylphosphonate(42 g, 88.4 mmol) was hydrogenated in ethanol in the presence of 6 g of10% Pd on charcoal to give (+)-diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-aminomethylphosphonate (24.5 g;75% yield; mp=143-144° C. (recrystallized from ligroin/MTBE); [α]_(D)²⁹+11.04° (c=1.714, CHCl₃)].

(+)-Diethyl α-(3,5-di-tert-butyl-4-hydroxyphenyl)-aminomethylphosphonate(11 g, 29.6 mmol) and pyridine-4-carboxaldehyde (6.35 g, 59.3 mmol) in125 ml of MeOH were reacted with NaBH₃CN (5.6 g, 89 mmol) in the samemanner as described for the (−) enantiomer. Column chromatography onsilicagel (95/5 CHCl₃/MeOH) gave (+)diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(4-picolyl)-aminomethylphosphonate(12 g, 87% yield; mp=67-70° C.; [α]_(D) ²¹+43.03° (c=1.984, CHCl₃)].

EXAMPLE 17 Enantiomers of Diethylα-(3,5-di-tert-Butyl-4-hydroxyphenyl)-N-(3-picolyl)-aminomethylphosphonate

a) In the same manner as described in example 16, (+)-diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)aminomethylphosphonate (1 g, 2.7mmol) and pyridine-3-carboxaldehyde (0.43 g, 4 mmol) were reacted withNaBH₃CN (0.34 g, 5.4 mmol) in MeOH for 5 hours at room temperature toyield after trituration in petroleum ether(+)-diethyl-α-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(3-picolyl)-aminomethylphosphonate(1 g, 80% yield; mp=116-119° C.; [α]_(D) ²³+42.88° (c=1.614, CHCl₃)].

b) Respectively,(−)-diethyl-α-(3,5-di-tert-butyl-4-hydroxyphenyl)-aminomethylphosphonate(1 g, 2.7 mmol) and pyridine-3-carboxaldehyde (0.43 g, 4 mmol) werereacted with NaBH₃CN (0.34 g, 5.4 mmol) in MeOH to give(−)-diethyl-α-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(3-picolyl)aminomethylphosphonate(0.7 g, 56%; mp=118-120° C.).

EXAMPLE 18 Enantiomers of Diethylα-(3,5-Dimethoxy-4-hydroxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate

The enantiomers of a racemic mixture were separated by preparative HPLCon Chiralcel OD and isocratic elution with hexane/ethanol (9:1), UVdetection at 254 run. Baseline separation was achieved, and the contentsof both peaks were evaporated to white solids in which none of the otherisomer could be detected by analytical HPLC.

First peak: retention time 18 min, [α]_(D) ²⁰−7.4° (c=0.244% w/v, EtOH);Second peak: retention time 34 min, [α]_(D) ²⁰+8.3° (c=0.255% w/V,EtOH).

The structures of both enantiomers were confirmed by NMR and MSspectroscopies and elemental analysis.

Elemental analysis: C₁₈H₂₅N₂O₆P % Calc. C 54.54 H 6.36 N 7.07 (+)Enantiomer: mp: 153-157° % Found C 53.85 H 6.22 N 6.81 (−) Enantiomer:mp: 155-158° % Found C 54.25 H 6.24 N 6.94

EXAMPLE 19 Enantiomers of Diethylα-(3,5-di-tert-Butyl-4-hydroxyphenyl)-N-(3-phenylpropyl)aminomethylphosphonate

a)(−)-Diethyl-α-(3,5-di-tert-butyl-4-hydroxyphenyl)-aminomethylphosphonate(1.7 g, 4.5 mmol) and 3-phenylpropionaldehyde (0.6 g, 4.5 mmol) in 20 mlof absolute methanol were stirred under nitrogen, at room temperaturefor 30 min. NaBH₃CN (0.3 g, 4.5 mmol) dissolved in 10 ml of methanol wasadded and the mixture was allowed to react at room temperature foranother hour. The reaction mixture was evaporated to dryness and theresidue dissolved in CH₂Cl₂. The organic phase was washed with water,then dried over MgSO₄. Column chromatography with 98/2 CHCl₃/MeOH aseluent gave(−)-diethyl-α-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(3-phenylpropyl)aminomethylphosphonate[1.2 g; 56% yield; [α]_(D) ²⁵+33.1° (c=2.055, CHCl₃)].

b) (+) Diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-aminomethylphosphonate (1.2 g, 3.2mmol) and 3-phenylpropionaldehyde (0.4 g, 3.2 mmol) in 20 ml of absolutemethanol were reacted in the same manner with NaBH₃CN (0.2 g, 3.2 mmol)in 10 ml of methanol to yield after column chromatography, (+) diethylα-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(3-phenylpropyl)-aminomethylphosphonate[0.94 g; 61% yield; [α]_(D) ²⁵+31.1° (c=1.930, CHCl₃)].

c)—The structures of both enantiomers were confirmed by IR, NMR and MS.They were separated by analytical HPLC on Chiralpak AD and isocraticelution with hexane/2-propanol (9:1/v:v).

EXAMPLE 20 Diisopropylα-(3,5-Dimethoxy-4-hydroxyphenyl)-N-(3-pyridyl-aminomethylphosphonate

Diisopropyl phosphite (3.3 g, 20 mmol) was added to 2.58 g (10 mmol) of3,5-dimethoxy-4-hydroxybenzaldehyde N-(3-pyridyl) imine dissolved in 15ml toluene and the mixture was refluxed for 17 h. The solvent and theexcess of diisopropyl phosphite were evaporated and the residue waspurified by column chromatography (9/1 CH₂Cl₂/MeOH) andrecrystallisation from a mixture of EtOH/AcOEt to give 1.56 g (37%) of awhite solid, mp=157-160°.

MS (m/e)=424: M⁺, 259 (100%): M⁺ —PO₃iPr₂; NMR (CDCl₃): δ=8.08. 7.96,7.03 and 6.84 (4m, 1H each): aromatic H, 3-pyridyl, 6.69 (d, J=2 Hz,2H): aromatic H, substituted phenyl, 5.8 (broad, 1H): OH, 4.82 (d×d, 1H,J=7 and 10 Hz): N—H, 4.55 (d×d, 1H, J=7 and 23 Hz): CH—PO₃iPr₂,4.75-4.65 and 4.55-4.45 (2m, 2H total): P—O—CH—(CH₃)₂, 3.86 (s, 6H): OCH₃, 1.34, 1.28, 1.24 and 0.9: (4d, J=7 Hz): P—O—CH—(CH ₃)₂.

EXAMPLE 21 Diisopropylα-(4-Hydroxy-3-methoxy-5-methylphenyl)-N-(3-pyridyl)-amino-methylphosphonate

A mixture of 1.9 g (11 mmol) of 4-hydroxy-3-methoxy-5-methylbenzaldehyde(mp=98-100°) and 1.08 g (11 mmol) of 3-aminopyridine dissolved in 15 mltoluene and a catalytic amount of p-toluenesulfonic acid (ca. 5 mg)contained in a flask connected to a Dean Stark apparatus was refluxedfor 15 h. The solution was evaporated to dryness to give 2.7 g (100%) ofthe crude imine.

Diisopropyl phosphite (5.48 g, 33 mmol) was added to 2.77 g (11 mmol) ofthe above imine dissolved in 20 ml THF and the mixture was refluxed for24 h. The solvent and the excess of diisopropyl phosphite wereevaporated and the residue was purified by column chromatography (95/5CHCl₃/MeOH) and recrystallisation from a mixture of petroleumether/CH₂Cl₂ to yield 1.9 g (43%) of a white solid, mp=123-124°.

MS (m/e)=408: M⁺, 243 (100%): M⁺ —PO₃iPr₂ NMR (CDCl₃): δ=8.07, 7.95,7.02 and 6.84 (4m, 1H each): aromatic H, 3-pyridyl, 6.83-6.81: (m, 2H):aromatic H, substituted phenyl, 5.8 (s, 1H): OH, 4.78 (d×d, 1H, J=7.5and 10 Hz): N—H, 4.30 (d×d, 1H, J=7.5 and 23 Hz): CH—PO₃iPr₂, 4.73-4.65and 4.48-4.40 (2m, 2H total): P—O—CH—(CH₃)₂, 3.85 (s, 3H): OCH ₃, 2.22(s, 3H): CH ₃, 1.33, 1.26, 1.24 and 0.96: (4d, J=7 Hz): P—O—CH—(CH ₃)₂.

EXAMPLE 22 Diisopropylα-(3-n-Butyl-4-hydroxy-5-methoxyphenyl)-N-(3-pyridyl)-amino-methylphosphonate

A mixture of 6.1 g (30 mmol) of3-n-butyl-4-hydroxy-5-methoxybenzaldehyde and 2.76 g (30 mmol) of3-aminopyridine dissolved in 50 ml toluene and a catalytic amount ofp-toluenesulfonic acid (ca. 5 mg) contained in a flask connected to aDean Stark apparatus was refluxed for 16 h. The solution was evaporatedto dryness to give 7.8 g (94%) of the crude imine.

Diisopropyl phosphite (4.20 g, 25 mmol) was added to 2.4 g (8 mmol) ofthe above imine dissolved in 30 ml THF and the mixture was refluxed for24 h. The solvent and the excess of diisopropyl phosphite wereevaporated and the residue was purified by column chromatography (95/5CHCl₃/MeOH) and recrystallisation from a mixture of petroleumether/CH₂Cl₂ to yield 1.9 g (43%) of a white solid, mp=142-144°.

MS (m/e)=450: M⁺, 285 (100%): M⁺ —PO₃iPr₂; NMR (CDCl₃): δ=8.07, 7.95,7.0 and 6.84 (4m, 1H each): aromatic H, 3-pyridyl, 6.83-6.80: (m, 2H):aromatic H, substituted phenyl, 5.8 (s, 1H): OH, 4.74 (d×d, 1H, J=7.5and 10 Hz): N—H, 4.54 (d×d, 1H, J=7.5 and 23 Hz): CH—PO₃iPr₂, 4.75-4.65and 4.50-4.40 (2m, 2H total): P—O—CH—(CH₃)₂, 3.85 (s, 3H): OCH ₃, 2.60(t, 2H), 1.5 (m, 2H), 1.31 (m, 2H) and 0.90 (t, 3H): n-Bu, 1.33, 1.26,1.24 and 0.94: (4d, J=7 Hz): P—O—CH—(CH ₃)₂.

EXAMPLE 23 Diethylα-(3,5-Dimethoxy-4-hydroxyphenyl)-N-methyl-N-(3-picolyl)aminomethylphosphonate

A mixture of 3.0 g (16.5 mmol) of syringaldehyde, 2.03 g (16.6 mmol) ofN-methyl-3-picolylamine and 2.3g (16.6 mmol) diethyl phosphite dissolvedin 15 ml toluene and a catalytic amount of p-toluenesulfonic acid (ca. 5mg) contained in a flask connected to a Dean Stark apparatus wasrefluxed for 2 h. The solution was evaporated and the residue waspurified by column chromatography (95/5 CHCl₃/MeOH) to yield 3.2 g (46%)of a yellow oil.

MS (m/e)=287: M⁺ —PO₃Et₂; NMR (CDCl₃): δ=8.55, 8.51, 7.72 and 7.27 (4m,1H each): aromatic H, 3-picolyl, 6.73 (d, 2H): aromatic H, substitutedphenyl, 5.8 (broad, 1H): OH, 4.25, 3.94 and 3.7 (3m, 4H): P—O—CH ₂—CH₃,3.89 (d, J=23 Hz, 1H): CH—PO₃Et₂, 3.9 and 3.4 (2d, 2H): N(CH₃)—CH ₂—Py,3.91 (s, 6H): OCH ₃, 2.41 (s, 3H): N(CH ₃—CH₂—Py, 1.39 and 1.08 (2t, J=7Hz, 6 H): P—O—CH₂CH ₃.

EXAMPLE 24 Diisopropylα-(4-Hydroxy-3-methoxy-5-methylphenyl)-N-methyl-N-(3-picolyl)-aminomethylphosphonate

A mixture of 2.0 g (12 mmol) of4-hydroxy-3-methoxy-5-methylbenzaldehyde, 1.8 g (13.2 mmol) ofN-methyl-3-picolylamine and 2.2 g (13.2 mmol) diisopropyl phosphitedissolved in 15 ml toluene and a catalytic amount of p-toluenesulfonicacid (ca. 2 mg) contained in a flask connected to a Dean Stark apparatuswas refluxed for 2 h. The solution was evaporated and the residue waspurified by column chromatography (95/5 CHCl₃/MeOH) to yield 2.1 g (40%)of a yellow oil.

MS (m/e)=271 (100%): M⁺ —PO₃iPr₂; NMR (CDCl₃): δ=8.54, 8.50, 7.72 and7.24 (4m, 1H each): aromatic H, 3-picolyl, 6.97 and 6.77 (2 m, 2H):aromatic H. substituted phenyl, 5.75 (broad, 1H): OH, 4.86-4.78 and4.51-4.42 (2m, 2H total): P—OH—(CH₃)₂, 3.84 (d, J=24 Hz, 1H):CH—PO₃iPr₂, 3.97 and 3.34 (2d, J=13,5 Hz, 2H): N(CH₃)—CH ₂—Py, 3.91 (s,3H): OCH ₃, 2.36 (s, 3H): CH ₃, 2.26 (s, 3H): N(CH ₃)—CH₂—Py, 1.39,1.37, 1.21 and 0.83 (4d, J=7 Hz, 12H): P—O—CH(CH ₃)₂.

The following compounds may also be obtained in an analogous manner toExamples 1 to 24:

Diethylα-(4-hydroxy-3-methoxy-5-n-propylphenyl)-N-(3-pyridyl)-aminomethylphosphonate;

Diisopropylα-(4-hydroxy-3-methoxy-5-n-propylphenyl)-N-(3-pyridyl)-aminomethylphosphonate;

Diethylα-(3-i-butyl-4-hydroxy-5-methoxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate;and

Diisopropylα-(3-i-butyl-4-hydroxy-5-methoxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate.

Table 1 lists the physicochemical data of compounds of formula (I) thatwere prepared by the methods illustrated by examples 1-24 of thisapplication. These methods are disclosed in EP 0 559 079A (correspondingto the U.S. Pat. No. 5,424,303).

TABLE 1 Aminophosphonates of formula (I) (I)

Cpd X¹ X² X³ Z A R mp (° C.) Microanalysis  1 tBu tBu H H 3-pyridyl Me168-170 C₂₂H₃₃N₂O₄P  2 tBu tBu H H 3-pyridyl Et 155-156 C₂₄H₃₇N₂O₄P  3tBu tBu H H 3-pyridyl iPr 135-137 C₂₆H₄₁N₂O₄P  4 tBu tBu H H 3-pyridylnPr 133-135 C₂₆H₄₁N₂O₄P  5 tBu tBu H H 3-pyridyl nBu 112-114 C₂₈H₄₅N₂O₄P 6 tBu tBu H H 4-picolyl Me 120-122 C₂₃H₃₅N₂O₄P  7 tBu tBu H H 4-picolylEt 87-91 C₂₅H₃₉N₂O₄P  8 tBu tBu H H 4-picolyl iPr 126-128 C₂₇H₄₃N₂O₄P  9tBu tBu H H 4-picolyl nPr 108-110 C₂₇H₄₃N₂O₄P 10 tBu tBu H H 4-picolylnBu 60-61 C₂₉H₄₇N₂O₄P 11 tBu tBu H COMe 4-picolyl Et 160-162 C₂₇H₄₁N₂O₅P12 tBu tBu H H 5-(2-chloropyridyl) Et 124-126 C₂₄H₃₆ClN₂O₄P 13 tBu tBu HH 2-pyridyl Et 116-118 C₂₄H₃₇N₂O₄P 14 tBu tBu H H 4-pyridyl Et 116-119C₂₄H₃₇N₂O₄P 15 tBu tBu H H 3-picolyl Et 100-101 C₂₅H₃₉N₂O₄P 16 tBu tBu HH 2-picolyl Et 90-91 C₂₅H₃₉N₂O₄P 17 tBu tBu H H 2-(2-pyridyl)ethyl Et76-78 C₂₆H₄₁N₂O₄P 18 H H H H 3-pyridyl Et 210-212 C₁₆H₂₁N₂O₄P 19 OMe OMeH H 3-pyridyl Me 186-187 C₁₆H₂₁N₂O₆P 20 OMe OMe H H 3-pyridyl Et 181-183C₁₈H₂₅N₂O₆P 21 OMe OMe H H 3-pyridyl iPr 157-160 C₂₀H₂₉N₂O₆P 22 OMe OMeH H 2-picolyl Et oil C₁₉H₂₇N₂O₆P 23 OMe OMe H H 3-picolyl Et  99-101C₁₉H₂₇N₂O₆P 24 OMe OMe H H 4-picolyl Et 125-127 C₁₉H₂₇N₂O₆P 25 OMe H H H3-pyridyl Et 171-173 C₁₇H₂₃N₂O₅P 26 OEt H H H 3-pyridyl Et 185-187C₁₈H₂₅N₂O₅P 27 OMe OMe Me H 3-pyridyl Et 134-136 C₁₉H₂₇N₂O₆P 28 Me Me HH 3-pyridyl Et 176-178 C₁₈H₂₅N₂O₄P 29 OMe OMe H H 2-pyridyl Et 163-165C₁₈H₂₅N₂O₆P 30 OMe OMe H H 4-pyridyl Et 172-174 C₁₈H₂₅N₂O₆P 31 OMe OMe HH 3-pyridyl nPr 142-143 C₂₀H₂₉N₂O₆P 32 OMe OMe H H 3-pyridyl nBu 158-160C₂₂H₃₃N₂O₆P 33 OMe NO₂ H H 3-pyridyl Et 212-213 C₁₇H₂₂N₃O₇P 34 OMe OMe HH 5-(2-chloropyridyl) Et 193-195 C₁₈H₂₄ClN₂O₆P 35 OMe OMe H H5-(2-methoxypyridyl) Et 135-137 C₁₉H₂₇N₂O₇P 36 OMe OMe H H3-(2-methylpyridyl) iPr 148-150 C₂₁H₃₁N₂O₆P 37 OMe OMe H H5-(2-methylpyridyl) Et 189-190 C₁₉H₂₇N₂O₆P 38 OMe OMe H H5-(2-methylpyridyl) iPr 150-152 C₂₁H₃₁N₂O₆P 39 Me t-Bu H H 3-pyridyl Et90-91 C₂₁H₃₁N₂O₄P 40 iPr iPr H H 3-pyridyl Et 174-176 C₂₂H₃₃N₂O₄P 41 sBusBu H H 3-pyridyl Et 140-141 C₂₄H₃₇N₂O₄P 42 Et Et H H 3-pyridyl Et170-171 C₂₀H₂₉N₂O₄P 43 OMe Me H H 3-pyridyl Et 164-166 C₁₈H₂₅N₂O₅P 44OMe Me H H 3-pyridyl iPr 123-125 C₂₀H₂₉N₂O₅P 45 OMe nBu H H 3-pyridyl Et133-134 C₂₁H₃₁N₂O₅P 46 OMe nBu H H 3-pyridyl iPr 142-144 C₂₃H₃₅N₂O₅P 47OMe Me H H 3-picolyl Et  99-101 C₁₉H₂₇N₂O₅P 48 OMe Me H H 3-picolyl iPr85-86 C₂₁H₃₁N₂O₅P 49 OMe Me H H 4-picolyl Et 129-130 C₁₉H₂₇N₂O₅P 50 OMeMe H H 4-picolyl iPr 138-141 C₂₁H₃₁N₂O₅P 51 Me Me H H 3-pyridyl iPr169-171 C₂₀H₂₉N₂O₄P 52 OEt H H H 3-pyridyl iPr 192-194 C₂₀H₂₉N₂O₅P 53OEt Me H H 3-pyridyl Et 172-173 C₁₉H₂₇N₂O₅P 54 OEt Me H H 3-pyridyl iPr177-178 C₂₁H₃₁N₂O₅P 55 OEt OEt H H 3-pyridyl Et 130-132 C₂₀H₂₉N₂O₆P 56OEt OEt H H 3-pyridyl iPr 149-150 C₂₂H₃₃N₂O₆P 57 OMe Et H H 3-pyridyl Et139-141 C₁₉H₂₇N₂O₅P 58 OMe Et H H 3-pyridyl iPr 146-148 C₂₁H₃₁N₂O₅P 59OMe OEt H H 3-pyridyl Et 156-157 C₁₉H₂₇N₂O₆P 60 OMe OEt H H 3-pyridyliPr 159-160 C₂₁H₃₁N₂O₆P 61 OMe OMe H Me 3-picolyl Et oil *NMR and MS 62OMe OMe H Me 3-picolyl iPr oil *NMR and MS 63 OMe Me H Me 3-picolyl Etoil *NMR and MS 64 OMe Me H Me 3-picolyl iPr oil *NMR and MS 65 OMe OMeH H 3-pyridyl Et 153-157 **C₁₈H₂₅N₂O₆P 66 OMe OMe H H 3-pyridyl Et155-158 ***C₁₈H₂₅N₂O₆P 67 OMe OMe H H phenyl Et 143-146 C₁₉H₂₆NO₆P 68OMe OMe H H phenyl iPr 144-146 C₂₁H₃₀NO₆P 69 OMe Me H H5-(2-methylpyridyl) Et 154-155 C₁₉H₂₇N₂O₅P 70 OMe Me H H5-(2-methylpyridyl) iPr 149-150 C₂₁H₃₁N₂O₅P 71 OMe Me H H3-(2-methylpyridyl) Et 150-152 C₁₉H₂₇N₂O₅P 72 OMe Me H H3-(2-methylpyridyl) iPr 148-150 C₂₁H₃₁N₂O₅P 73 OMe OMe H H3-(2-methylpyridyl) Et 146-148 C₁₉H₂₇N₂O₆P *Identified by NMR and MSspectroscopies **(+) Enantiomer of Compound 20 ***(−) Enantiomer ofCompound 20 (I)

Cpd X¹ X² X³ (B)_(n) Z R mp (° C.) Microanalysis H OCH₂ bond H Et 98-99C₁₇H₂₁N₂O₅ OMe OMe H CH═CH H Et foam *NMR and MS OMe OMe H CH₂—CH₂ H Et134-138 C₂₀H₂₉N₂O₆P *Identified by NMR and MS spectroscopies

Biological Data

In vitro Data—The compounds of formula (I) were tested for lowering theproduction of Lp(a) in primary cultures of Cynomolgus hepatocytesaccording to the assays described below. Two incubation times were used:4 h for Assay 1 and 24 h for Assay 2.

Protocol—Hepatocytes were isolated from livers of adult Cynomolgusmonkeys by the two-step collagenase perfusion method according to C.Guguen-Guillouzo and A. Guillouzo “Methods for preparation of adult andfetal hepatocytes” p.1-12 in “Isolated and Cultured Hepatocytes”, leseditions Inserm Paris and John Libbey Eurotext London (1986).

The viability of cells was determined by Trypan blue staining. The cellswere then seeded at a density of 1.5-2.10⁵ viable cells per 2 cm² in 24well tissue culture plates in a volume of 500 μl per well of Williams Etissue culture medium containing 10% fetal calf serum. Cells wereincubated for 4-6 hours at 37° C. in a CO₂ incubator (5% CO₂) in thepresence of 20 μM of the test compounds dissolved in ethanol. Four wellswere used for each compound. Nicotinic acid and steroid hormones wereused as references to validate the assay system since they are known todecrease Lp(a) in man. Control cells were incubated in the presence ofethanol only.

The amount of Lp(a) secreted in culture medium was directly assayed byELISA using a commercially available kit. Cells were washed and lysed asdescribed by A. L. White et al, Journal of Lipid Research vol 34, p.509-517, (1993) and the cellular content of Lp(a) was assayed asdescribed above.

Changes in Lp(a) concentration in culture medium are given as thepercentage of value measured for the control plates at 4 h (Assay 1) or24 h (Assay 2).

Results—Assay 1: compounds 2, 7, 11, 15, 16, 18 and 20 were found tochange the concentrations of Lp(a) in the culture medium in the rangefrom −12 to −34%.

Assay 2: compounds 1, 2, 3, 5, 7, 11, 13, 15, 17, 19, 20, 21, 26 to 29,32, 34 to 52, 57 to 60, 65 and 66 were found to change theconcentrations of Lp(a) in the culture medium in the range from −7 to−37%.

In Vivo Data—Study Protocol—Male cynomolgus monkeys weighing between 3and 7 kg were divided into groups of 3 to 4 animals each. Prior totreatment their plasma Lp(a) levels were followed over a two monthperiod to ascertain a constant baseline value. Test compounds were givenorally by gavage at the dose of 25 mg/kg/day for 4 weeks and Lp(a) wasmeasured at day 28. At the end of the dosing period, animals weremaintained for a treatment free period of 4 weeks, whereupon theirplasma Lp(a) levels returned to pretreatment levels. This controlprovided proof that the decrease in Lp(a) measured was caused by thepharmacological activity of the test compounds.

Results—At Days −7 and 28, after an overnight fast, blood samples werecollected on EDTA and Lp(a) was measured by the highly sensitive andspecific ELISA test. Results (mean of 3-4 values of each group) wereexpressed as % of predose (Day −7). Selected compounds of formula (I)were tested under the experimental conditions to investigate theirpharmacological activity in vivo.

The compounds No 1, 2, 3, 7, 15, 17, 19, 20, 21, 27, 28, 32, 39, 44 and52 lower plasma Lp(a) in the range of −13% to −51% (value measured atDay 28, % change from predose at Day −7).

The compounds of Formula (I) have therefore a therapeutic potential forthe treatment of the following diseases where Lp(a) is associated withaccelerated atherosclerosis, abnormal proliferation smooth muscle cellsand increased thrombogenesis: coronary heart disease, peripheral arterydisease: intermittent claudication, extracranial carotidatherosclerosis, stroke, restenosis after angioplasty andatherosclerosis occuring after heart transplant. The primary indicationsof these compounds would be the treatment of the diseases mentionedabove.

What is claimed is:
 1. A process for preparing a compound of formula(Ia):

wherein X¹ is H, C₍₁₋₃₎alkyl, hydroxy or C₍₁₋₄₎alkoxy; X² is C₍₁₋₃₎alkylor C₍₁₋₄₎alkoxy; X³ is H or C₍₁₋₄₎alkyl; R¹, R², which may be identicalor different, are H or C₍₁₋₃₎alkyl; B is CH₂CH₂, CH═CH, or CH₂; n iszero or 1; Z is H; m is an integer from 1 to 5; X⁴ is H, or C₍₁₋₈₎alkyl,C₍₁₋₈₎alkoxy or halo; and the pyridyl ring is attached by the ringcarbon α- or β- to the nitrogen (2- or 3-pyridyl); which processcomprises (a) treating an amine of formula (VII)

in which B, R¹, R², X¹, X², X³ and n are as hereinbefore defined; withan aldehyde of formula (IX):

in which m and X⁴ are as hereinbefore defined; under reductive aminationconditions.
 2. A process for preparing an individual enantiomer of anaminophosphonate of formula (Ia) as defined in claim 1 which processcomprises treating either of the (+) or (−) enantiomer of theα-substituted aminomethylphosphonate of formula (X):

in which B, R¹, R², X¹, X², X³ and n are as defined in claim 1; with analdehyde of formula (IX):

in which m and X⁴ are as defined in claim 1; under reductive aminationconditions.
 3. A process as claimed in claim 2 in which the reaction iscarried out in the presence of sodium cyanoborohydride in an alcoholicsolvent, preferably methanol, at a pH between 3 to 6 and at atemperature between 0° C. and 25° C.
 4. A compound selected from thegroup consisting of: diisopropylα-(4-hydroxy-3-methoxy-5-methylphenyl)-N-(3-pyridyl)-aminomethylphosphonate;dimethylα-(3,5-dimethoxy-4-hydroxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate;diethylα-(3,5-dimethoxy-4-hydroxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate;diisopropylα-(3,5-dimethoxy-4-hydroxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate;diethylα-(3,5-dimethyl-4-hydroxyphenyl)-N-(3-pyridyl)-aminomethylphosphonate;and dimethylα-(3-methyl-4-hydroxyphenyl-5-tert-butyl)-N-(3-pyridyl)-aminomethylphosphonate.5. A pharmaceutical composition comprising a compound of claim 4 and apharmaceutically acceptable carrier or excipient.
 6. The compound ofclaim 4, wherein said compound is diisopropylα-(4-hydroxy-3-methoxy-5-methylphenyl)-N-(3-pyridyl)-aminomethylphosphonate.7. The compound of claim 6, wherein said compound is (+)diisopropylα-(4-hydroxy-3-methoxy-5-methylphenyl)-N-(3-pyridyl)-aminomethylphosphonate.8. The compound of claim 6, wherein said compound is (−)diisopropylα-(4-hydroxy-3-methoxy-5-methylphenyl)-N-(3-pyridyl)-aminomethylphosphonate.